Genes and derived amino acid sequences of S-layer proteins from mesophilic, thermophilic, and extremely thermophilic methanococci

Cells of methanococci are covered by a single layer of protein subunits (S-layer) in hexagonal arrangement, which are directly exposed to the environment and which cannot be stabilized by cellular components. We have isolated S-layer proteins from cells of Methanococcus vannielii ( T(opt.)=37 degrees C), Methanococcus thermolithotrophicus ( T(opt.)=65 degrees C), and Methanococcus jannaschii ( T(opt.)=85 degrees C). The primary structure of the S-layer proteins was determined by sequencing the corresponding genes. According to the predicted amino acid sequence, the molecular masses of the S-layer proteins of the different methanococci are in a small range between 59,064 and 60,547 Da. Compa…

Termite Gut Symbiotic Archaezoa Are Becoming Living Metabolic Fossils

ABSTRACT Over the course of several million years, the eukaryotic gut symbionts of lower termites have become adapted to a cellulolytic environment. Up to now it has been believed that they produce nutriments using their own cellulolytic enzymes for the benefit of their termite host. However, we have now isolated two endoglucanases with similar apparent molecular masses of approximately 36 kDa from the not yet culturable symbiotic Archaezoa living in the hindgut of the most primitive Australian termite, Mastotermes darwiniensis . The N-terminal sequences of these cellulases exhibited significant homology to cellulases of termite origin, which belong to glycosyl hydrolase family 9. The corre…

Cell Wall Structures of Mesophilic, Thermophilic and Hyperthermophilic Archaea

Aerobic Endospore-forming Bacteria and Soil Invertebrates

The intestinal microbiotas of only a few soil invertebrates such as collembola (springtails), earthworms, nematodes, isopods (woodlice and pill bugs), millipedes and termites have been studied by classical and molecular methods in the last decades. It became obvious that these lower members of the soil biota harbour complex microbial communities that sometimes reach counts of 1011 cells ml−1 in their intestinal tracts. The gut microbiotas of the soil fauna include a variety of microorganisms from all three domains of life (Bacteria, Archaea, Eucarya). The bacterial species can mainly be assigned to the Gram-positive phyla Firmicutes and Actinobacteria as well as the Gram-negative phyla Prot…

A novel ether core lipid with H-shaped C80-isoprenoid hydrocarbon chain from the hyperthermophilic methanogen Methanothermus fervidus.

Abstract A new ether lipid core (designated as FU) was found in Methanothermus fervidus total lipid. Comparison with caldarchaeol showed lower mobility of FU on TLC and smaller molecular weight (m/z 1298) by 2 mass units on FAB-MS. Treatment of FU with HI followed by displacement with silver acetate afforded long chain alcohol acetate (ROAc), which was further saponified with mild alkali to its free alcohol (ROH). ROH is the long chain alcohol prepared from FU. The molecular weights of ROAc and ROH were shown by MS to be 1354 and 1186, respectively. These results suggested that the molecular formula of ROH was C80H162O4, and ROH had four hydroxyl groups, and one molecule of ROH was bound wi…

The development of species-specific probes for a quantitative estimation of non-cultured bacteria in groundwater samples

The aim of this study was to quantify different bacteria in drinking water production wells that supply the city of Mainz (capital of Rhineland-Palatinate, Germany). Knowledge of the counts of individual microorganisms is important for the appraisal of the hygienic and sensory quality of drinking water. Specific 16S rRNA probes were designed for 14 previously identified but non-cultured bacterial species ( Ferribacterium limneticum , Hydrogenophaga sp., Methylotenera sp., Janthinobacterium sp., Pseudomonas sp., Rhizobium selenireducens , Rhodoferax sp., Nitrosomonadaceae bacterium, Thauera sp. and five strains which could not be identified to species level). With fluorescence in situ hybrid…

Occurrence of rhizobia in the gut of the higher termite Nasutitermes nigriceps

Wood-eating termites feed on a diet highly deficient in nitrogen. They must complement their diet with the aid of nitrogen-fixing bacteria. Nitrogen fixation in the gut has been demonstrated, but information about nitrogen-fixing bacteria in pure culture is scarce. From the higher termite Nasutitermes nigriceps the symbiotic bacterial strain M3A was isolated, which thrives in the hindgut contents. The Gram-negative strain exhibited similarities to the species of the genus Ensifer (including Sinorhizobium) on the basis of morphological and physiological/biochemical features. The 16S rRNA gene analysis showed the highest sequence similarity of the isolate M3A to Ensifer adhaerens (>99%; ATCC …

Molecular Identification of Lactic Acid Bacteria Occurring in Must and Wine

A specifically amplified polymorphic DNA-polymerase chain reaction (SAPD-PCR), a molecular fingerprinting method based on the amplification of specific gene sequences, was applied in order to allow a rapid identification of lactic acid bacteria (LAB) occurring in must and wine. The applicability of this method was confirmed with isolated strains from different wine samples from the German wine growing region Palatinate. In addition, the formation of biogenic amines by the isolated strains was studied. More than half of the bacterial isolates from 50 red and white wine samples were able to produce biogenic amines. General health concerns related to biogenic amines in must and wine underline …

Spirochaeta coccoides sp. nov., a novel coccoid spirochete from the hindgut of the termite Neotermes castaneus.

ABSTRACT A novel spirochete strain, SPN1, was isolated from the hindgut contents of the termite Neotermes castaneus . The highest similarities (about 90%) of the strain SPN1 16S rRNA gene sequence are with spirochetes belonging to the genus Spirochaeta , and thus, the isolate could not be assigned to the so-called termite clusters of the treponemes or to a known species of the genus Spirochaeta . Therefore, it represents a novel species, which was named Spirochaeta coccoides . In contrast to all other known validly described spirochete species, strain SPN1 shows a coccoid morphology and is immotile. The isolated strain is obligately anaerobic and ferments different mono-, di-, and oligosacc…

Proteinaceous Surface Layers ofArchaea: Ultrastructure and Biochemistry

The cell walls of the Archaea are composed of different polymers such as glutaminylglycan, heterosaccharide, methanochondroitin, pseudomurein, protein, glycoprotein, or glycocalyx. The S-layer glycoprotein of Halobacterium salinarum was the first glycoprotein discovered in bacteria and archaea. Initially, the novel cell wall structures were viewed as curiosities, and their taxonomic significance was not realized until the concept of the Archaea was published. At this time, the results of cell wall studies supported the new view of the phylogeny of the Bacteria and Archaea. Many archaea possess proteinaceous surface layers (S layers), which form two-dimensional regular arrays. The chemical s…

Allelic variants of hexose transporter Hxt3p and hexokinases Hxk1p/Hxk2p in strains ofSaccharomyces cerevisiaeand interspecies hybrids

The transport of sugars across the plasma membrane is a critical step in the utilization of glucose and fructose by Saccharomyces cerevisiae during must fermentations. Variations in the molecular structure of hexose transporters and kinases may affect the ability of wine yeast strains to finish sugar fermentation, even under stressful wine conditions. In this context, we sequenced and compared genes encoding the hexose transporter Hxt3p and the kinases Hxk1p/Hxk2p of Saccharomyces strains and interspecies hybrids with different industrial usages and regional backgrounds. The Hxt3p primary structure varied in a small set of amino acids, which characterized robust yeast strains used for the p…

Pulsed-field gel electrophoresis for the discrimination of Oenococcus oeni isolates from different wine-growing regions in Germany

Reliable techniques are needed for the identification individual Oenococcus oeni strains with desirable flavor characteristics and to monitor the survival and contribution of inoculated and indigenous bacteria. Therefore, we investigated the suitability of pulsed-field gel electrophoresis (PFGE) for the discrimination of 65 O. oeni isolates from six different wine-producing regions in Germany. Among the restriction enzymes tested, genomic DNA digestions with Sfi I were most effective by displaying 56 (86%) different banding profiles. Our results underline the high capacity of PFGE for strain identification and differentiation. Cluster analysis of the DNA restriction patterns revealed no dis…

Chronological development of element concentrations in grapes during growth and ripeness and during fermentation of must determined by instrumental neutron-activation analyses

Abstract The chronological development of element concentrations during growth and ripeness of grapes described in the literature has only been concerned with the macro elements Mg, K, and Ca. Concentrations of trace elements in must are only described as a snapshot at the end of the ripeness. Therefore, the motivation for the present work was to accompany the growth and the ripening process of grapes successively by systematically determining element concentrations in grapes of Riesling and Cabernet Sauvignon by neutron-activation analyses. While for a number of elements, the concentrations in the grapes increased as a function of grape development (e.g., Na, K, Rb, Al), other concentratio…

Microbial Succession in Spontaneously Fermented Grape Must Before, During and After Stuck Fermentation

The microbial succession in spontaneously fermenting Riesling must was investigated from the beginning (pressing) until the end (sulphuring) of the fermentation in two harvest years (2008 and 2009) at a Moselle winery (Germany). In both years, the fermentation was interrupted by a stuck period. The length of the stuck period varied considerably (20 weeks in 2008 and one week in 2009). Different yeasts (Candida, Debaryomyces, Pichia, Hanseniaspora, Saccharomyces, Metschnikowia, Cryptococcus, Filobasidium and Rhodotorula) and bacteria (Gluconobacter, Asaia, Acetobacter, Oenococcus, Lactobacillus, Bacillus and Paenibacillus) were isolated successively by plating. The main fermenting organism w…

Zelloberflächenstrukturen der Bacteria und Archaea

Yeast Mixtures and Saccharomyces Hybrids: Suitable Tools for Performing More Sophisticated Must Fermentations

It can be concluded from the early development of viniculture that the yeast Saccharomyces cerevisiae is one of the oldest domesticated organisms. The wine producing techniques have been further developed empirically over generations. Despite this long time of wine production, our knowledge about the microbiological and biochemical background of the transformation of must into wine is relatively recent. Fundamental investigations about must transformation into wine were carried out at the end of the eighteenth and in the course of the nineteenth century. Today a relatively broad knowledge exists about the diversity of wine-related microorganisms. Nevertheless, sluggish or stuck fermentation…

Stuck at work? Quantitative proteomics of environmental wine yeast strains reveals the natural mechanism of overcoming stuck fermentation

During fermentation oenological yeast cells are subjected to a number of different stress conditions and must respond rapidly to the continuously changing environment of this harsh ecological niche. In this study we gained more insights into the cell adaptation mechanisms by linking proteome monitoring with knowledge on physiological behaviour of different strains during fermentation under model winemaking conditions. We used 2D-DIGE technology to monitor the proteome evolution of two newly discovered environmental yeast strains Saccharomyces bayanus and triple hybrid Saccharomyces cerevisiae × Saccharomyces kudriavzevii × S. bayanus and compared them to data obtained for the commercially a…

Degradation of dimeric lignin model compounds by aerobic bacteria isolated from the hindgut of xylophagous termites.

The capability of the intestinal flora from the gut of xylophagous termites of degrading lignin model compounds was investigated. Different dimeric lignin model compounds-degrading bacteria were obtained from the hindgut flora of Mastotermes darwiniensis FROGGATT, Reticulitermes santonensis FEYTAUD, Nasutitermes nigriceps HALDEMAN and Zootermopsis angusticollis HAGEN. In the presence of oxygen dimeric model compounds were degraded by all isolates. This indicates that the hindgut flora of termites is basically able to produce substrate for their host from aromatic extractives of wood.

The Potential of the Yeast Debaryomyces hansenii H525 to Degrade Biogenic Amines in Food

Twenty-six yeasts from different genera were investigated for their ability to metabolize biogenic amines. About half of the yeast strains produced one or more different biogenic amines, but some strains of Debaryomyces hansenii and Yarrowia lipolytica were also able to degrade such compounds. The most effective strain D. hanseniii H525 metabolized a broad spectrum of biogenic amines by growing and resting cells. Degradation of biogenic amines by this yeast isolate could be attributed to a peroxisomal amine oxidase activity. Strain H525 may be useful as a starter culture to reduce biogenic amines in fermented food.

Treponema isoptericolens sp. nov., a novel spirochaete from the hindgut of the termite Incisitermes tabogae.

A novel spirochaete, Treponema sp. strain SPIT5T, was isolated from hindgut contents of the drywood termite Incisitermes tabogae (Snyder). The cells of strain SPIT5T were motile, helical in shape, 0.4-0.5 microm in diameter and generally 12-20 microm long. The strain is obligately anaerobic and ferments different mono-, di- and oligosaccharides by forming ethanol as the main liquid fermentation end product. Furthermore, strain SPIT5T was able to grow anaerobically with yeast extract as sole carbon and energy source. Fastest growth was obtained at 30 degrees C, the temperature at which the termites were also grown. The optimum pH for growth was 7.2, with a range of pH 6.5-8.0. The cells poss…

Antioxidant capacity of phenolic compounds on human cell lines as affected by grape-tyrosinase and Botrytis-laccase oxidation.

Phenolic components (PCs) are well-known for their positive impact on human health. In addition to their action as radical scavengers, they act as activators for the intrinsic cellular antioxidant system. Polyphenol oxidases (PPOs) such as tyrosinase and laccase catalyze the enzymatic oxidation of PCs and thus, can alter their scavenging and antioxidative capacity. In this study, oxidation by tryosinase was shown to increase the antioxidant capacity of many PCs, especially those that lack adjacent aromatic hydroxyl groups. In contrast, oxidation by laccase tended to decrease the antioxidant capacity of red wine and distinct PCs. This was clearly demonstrated for p-coumaric acid and resverat…

Isolation of methanotrophic bacteria from termite gut.

The guts of termites feature suitable conditions for methane oxidizing bacteria (MOB) with their permanent production of CH4 and constant supply of O2 via tracheae. In this study, we have isolated MOB from the gut contents of the termites Incisitermes marginipennis, Mastotermes darwiniensis, and Neotermes castaneus for the first time. The existence of MOB was indicated by detecting pmoA, the gene for the particulate methane monooxygenase, in the DNA of gut contents. Fluorescence in situ hybridization and quantitative real-time polymerase chain reaction supported those findings. The MOB cell titer was determined to be 10(2)-10(3) per gut. Analyses of the 16S rDNA from isolates indicated clos…

Molecular Methods for Identification of Wine Microorganisms and Yeast Development

A prerequisite for the biochemical and physiological investigation of microorganisms is the isolation and management of pure cultures. Nevertheless, most of the environmental microorganisms are graded as “yet not cultivable” because the nutritional requirements are unknown or they could not be isolated due to the fact that fast-growing strains overgrow other microorganisms of a microbiota. In addition to plating techniques, isolation without cultivation and analysis of microbes could be performed by micromanipulation techniques or the application of optical tweezers followed by the utilization of PCR-based technologies.

Maintenance of Wine-Associated Microorganisms

A great variety of microorganisms growing on grapes, in must, or in wine have been isolated, which also have an influence on wine quality. They belong to acidtolerant microorganisms such as lactic acid bacteria, acetic acid bacteria, and yeasts. On grapes also molds can be found. The most important species for conversion of must into wine are the yeast Saccharomyces cerevisiae and the lactic acid bacterium Oenococcus oeni, which perform alcoholic and malolactic fermentation, respectively. Both species are used as starter cultures. A variety of techniques and media are available for the enrichment, culture, and preservation of these microorganisms. For selected species culture and preservati…

Biomethylation of Heavy Metals in Soil and Terrestrial Invertebrates

Heavy metals play a prominent role in the lives of all organisms. They can be essential, as in the cases of iron, manganese, nickel and copper, which are needed to obtain proper enzyme conformation and reactivity. Some heavy metals are toxic to organisms, such as mercury or cadmium. Often, these metals are rarely accessible in their inorganic form. After biological transformation into organometallic compounds, they exhibit increased toxicity and penetration into animal tissue is facilitated. The alkylation mechanisms of metals (especially mercury) performed by aquatic microorganisms have been well documented. The organometallic food chain from fish to humans has also been investigated. Howe…

Insights into the annotated genome sequence of Methanoculleus bourgensis MS2(T), related to dominant methanogens in biogas-producing plants

The final step of the biogas production process, the methanogenesis, is frequently dominated by members of the genus Methanoculleus. In particular, the species Methanoculleus bourgensis was identified to play a role in different biogas reactor systems. The genome of the type strain M. bourgensis MS2(T), originally isolated from a sewage sludge digestor, was completely sequenced to analyze putative adaptive genome features conferring competitiveness within biogas reactor environments to the strain. Sequencing and assembly of the M. bourgensis MS2(T) genome yielded a chromosome with a size of 2,789,773 bp. Comparative analysis of M. bourgensis MS2(T) and Methanoculleus marisnigri JR1 revealed…

Biology of Microorganisms on Grapes, in Must and in Wine

Diversity and Lignocellulolytic Activities of Cultured Microorganisms

The Intestinal Yeasts

Yeast isolates were obtained from the hindgut of the lower termites Mastotermis darwiniensis (Mastotermitidae), Zootermopsis angusticollis, Zootermopsis nevadensis (Hodotermitidae), Neotermes jouteli (Kalotermitidae), Reticulitermes santonensis, Heterotermes indicola (Rhinotermitidae) and the roach Cryptocercus punctulatus (Prillinger et al. 1996). The monosaccharide composition of the cell wall, the ubiquinone system, partial sequencing of 18S ribosomal DNA and the ultrastructure of septal pores indicate that most yeast species belonged to the Endomycetales. They were assigned to the genera Candida, Debaryomyces, Pichia and Sporothrix. Other species showed affinities to the Basidiomycetes …

Complete genome sequence of the strain Defluviitoga tunisiensis L3, isolated from a thermophilic, production-scale biogas plant.

An anaerobic, thermophilic bacterium belonging to the phylum Thermotogae was isolated from a rural, thermophilic biogas plant (54 degrees C) producing methane-rich biogas from maize silage, barley, cattle and pig manure. Here we report the first complete genome sequence of the Defluviitoga tunisiensis strain L3, an isolate from the family Thermotogaceae. The strain L3 encodes several genes predicted to be involved in utilization of a large diversity of complex carbohydrates including cellobiose and xylan for the production of acetate, hydrogen (H-2) and carbon dioxide (CO2). The genome sequence of D. tunisiensis L3 provides the basis for biotechnological exploitation of genetic determinants…

Methane oxidation in industrial biogas plants-Insights in a novel methanotrophic environment evidenced by pmoA gene analyses and stable isotope labelling studies.

Abstract A broad methanotrophic community consisting of 16 different operational taxonomic units (OTUs) was detected by particulate methane monooxygenase A (pmoA) gene analyses of reactor sludge samples obtained from an industrial biogas plant. Using a cloning-sequencing approach, 75% of the OTUs were affiliated to the group of type I methanotrophs (γ-Proteobacteria) and 25% to type II methanotrophs (α-Proteobacteria) with a distinct predominance of the genus Methylobacter. By database matching, half of the total OTUs may constitute entirely novel species. For evaluation of process conditions that support growth of methanotrophic bacteria, qPCR analyses of pmoA gene copy numbers were perfor…

Biosorption of copper by wine-relevant lactobacilli

Must and wine may be contaminated with elevated copper concentrations by the use of fungicides or in course of the vinification process. Hitherto only a few practicable and harmless procedures exist to reduce an excess of copper from must and wine. For this reason we investigated the biosorption of copper by eight wine-relevant Lactobacillus species. Both, living and heat-inactivated cells revealed a significant degree of Cu adsorption. It was shown that Cu binding correlated positively with an increasing pH value of the environment. The highest binding capacity of the tested lactic acid bacteria was found for L. buchneri DSM 20057 with a maximum of 46.17 μg Cu bound per mg cell in deionize…

Rapid isolation of single microbial cells from mixed natural and laboratory populations with the aid of a micromanipulator.

In order to facilitate the isolation of pure cultures from natural habitats we have developed a method for the isolation of single microbial cell clones from a mixed population, e.g. the flora of the termite gut, with the aid of a modern micromanipulator. The separated single prokaryotic or eukaryotic cells were grown after transfer in culture media or they were used for single cell PCR. The micromanipulator was also applied for the removal of nuclei from protozoa, of which the SSU rDNA was directly amplified.

Archaeal Cell Walls

Archaea represent the third domain of life with the Bacteria and Eukarya. They have developed different types of cell envelopes. Keywords: glutaminylglycan; heteropolysaccharide; methanochondroitin; pseudomurein; S-layer (glyco-)protein; Archaea; cell envelope

Bacterial Ectosymbionts which Confer Motility: Mixotricha paradoxa from the Intestine of the Australian Termite Mastotermes darwiniensis

Isolation of lactic acid-forming bacteria from biogas plants.

Direct molecular approaches provide hints that lactic acid bacteria play an important role in the degradation process of organic material to methanogenetic substrates in biogas plants. However, their diversity in biogas fermenter samples has not been analyzed in detail yet. For that reason, five different biogas fermenters, which were fed mainly with maize silage and manure from cattle or pigs, were examined for the occurrence of lactic acid-forming bacteria. A total of 197 lactic acid-forming bacterial strains were isolated, which we assigned to 21 species, belonging to the genera Bacillus, Clostridium, Lactobacillus, Pediococcus, Streptococcus and Pseudoramibacter-related. A qualitative m…

Stoffliche und energetische Nutzung von Weizenstroh zur Herstellung von Biopolymeren

Pyroglutamic Acid: A Novel Compound in Wines

Wine contains a complex mixture of nearly 1,000 compounds, which include sugars, acids, alcohols, ketones, aldehydes, esters, amino acids, amines, phenol carbonic acids, flavonoids, anthocyanidins and cathechins. Many of these compounds have an influence on the aroma and bouquet of wine. Identification of wine-related compounds is by far not complete. Recently, pyroglutamic acid has been detected as a so far overlooked novel compound in wine samples, which is described below.

Sugiyamaella mastotermitis sp. nov. and Papiliotrema odontotermitis f.a., sp. nov. from the gut of the termites Mastotermes darwiniensis and Odontotermes obesus

Two novel yeast species were isolated from the guts of two different termite species. A new member of the genus Sugiyamaella was isolated from the hindgut and nest material of the lower Australian termite Mastotermes darwiniensis. The second novel yeast species, isolated from the higher termite Odontotermes obesus, was identified as a member of the genus Papiliotrema. Both yeast species were able to hydrolyse xylan, methylumbelliferyl β-xylobiose and methylumbelliferyl β-xylotriose. The ability to debranch different hemicellulose side chains and growth without the addition of external vitamins was observed. A symbiotic role of the novel yeast species is indicated, especially in respect to x…

In vitro and in vivo sulfate reduction in the gut contents of the termite Mastotermes darwiniensis and the rose-chafer Pachnoda marginata.

Sulfate-reducing bacteria (SRB) from termites have been assigned to the genus Desulfovibrio. Desulfovibrio intestinalis lives in the gut of the Australian termite Mastotermes darwiniensis. For the first time we were able to enrich and identify a sulfate-reducing bacterium from the gut of the rose-chafer Pachnoda marginata, which showed the highest 16S rDNA sequence identity (93%) to Desulfovibrio intestinalis and Desulfovibrio strain STL1. Compared to Mastotermes darwiniensis (1x10(7) cells of SRB per ml gut contents), sulfate-reducing bacteria occurred in higher numbers in the gut contents of Pachnoda marginata reaching cell titers of up to 2x10(8) cells per ml gut contents. In vitro sulfa…

The first successful crystallization of a prokaryotic extremely thermophilic outer surface layer glycoprotein

AbstractMethanoihermus fervidusbelongs to the group of hyperthermophilic Archaea. The Archaea comprise organisms that live under environmental extremes, like high temperature, lowpH value or high salt concentration. The outer surface of the pseudomurein sacculi of the cells ofMethanothermus fervidusis covered by glycoprotein subunits (S-layer) directly exposed to the extreme environment. The elucidation of the crystal structure of this surface glycoprotein may provide important information on the survival strategies of these unusual micro-organisms. Before our investigations neither three-dimensional crystals have been obtained nor X-ray analyses were performed. Only electron microscopic an…

An Exemplary Model Study for Overcoming Stuck Fermentation during Spontaneous Fermentation with the Aid of a Saccharomyces Triple Hybrid

Sluggish or stuck fermentations cause significant financial losses for winemakers each year. In order to investigate the reasons for problems during spontaneous fermentation of Riesling must in a well-known German vineyard of the lower Moselle, yeast strains involved in must fermentation were identified during winemaking in the two years 2011 and 2012. Identification of the yeast isolates was performed by applying analyses of the ITS-1-5.8-ITS2 region and restriction fragment analyses of different gene sequences. It revealed that Saccharomyces (S.) bayanus and not Saccharomyces cerevisiae was the main fermenting yeast. Either S. bayanus finished the fermentation or led to stuck fermentation…

Influence of Laccase and Tyrosinase on the Antioxidant Capacity of Selected Phenolic Compounds on Human Cell Lines

Polyphenolic compounds affect the color, odor and taste of numerous food products of plant origin. In addition to the visual and gustatory properties, they serve as radical scavengers and have antioxidant effects. Polyphenols, especially resveratrol in red wine, have gained increasing scientific and public interest due to their presumptive beneficial impact on human health. Enzymatic oxidation of phenolic compounds takes place under the influence of polyphenol oxidases (PPO), including tyrosinase and laccase. Several studies have demonstrated the radical scavenger effect of plants, food products and individual polyphenols in vitro, but, apart from resveratrol, such impact has not been prove…

Properties of Halococcus salifodinae, an Isolate from Permian Rock Salt Deposits, Compared with Halococci from Surface Waters

Halococcus salifodinae BIpT DSM 8989T, an extremely halophilic archaeal isolate from an Austrian salt deposit (Bad Ischl), whose origin was dated to the Permian period, was described in 1994. Subsequently, several strains of the species have been isolated, some from similar but geographically separated salt deposits. Hcc. salifodinae may be regarded as one of the most ancient culturable species which existed already about 250 million years ago. Since its habitat probably did not change during this long period, its properties were presumably not subjected to the needs of mutational adaptation. Hcc. salifodinae and other isolates from ancient deposits would be suitable candidates for testing …

A search for beta-lactamase in chlamydiae, mycoplasmas, planctomycetes, and cyanelles: bacteria and bacterial descendants at different phylogenetic positions and stages of cell wall development.

Bacteria from different phylogenetic positions such as chlamydiae, mycoplasmas, planctomycetes and also endosymbiotic murein-containing cyanelles were investigated for the production of beta-lactamases. No beta-lactamase activity was found in bacteria lacking murein such as Chlamydia pneumoniae, Mycoplasma pneumoniae, Pirellula marina and Planctomyces maris. In the murein-containing cyanelles of Cyanophora paradoxa no beta-lactamase activity could be detected.

Genomic characterization of Defluviitoga tunisiensis L3, a key hydrolytic bacterium in a thermophilic biogas plant and its abundance as determined by metagenome fragment recruitment

The genome sequence of Defluviitoga tunisiensis L3 originating from a thermophilic biogas-production plant was established and recently published as Genome Announcement by our group. The circular chromosome of D. tunisiensis L3 has a size of 2,053,097bp and a mean GC content of 31.38%. To analyze the D. tunisiensis L3 genome sequence in more detail, a phylogenetic analysis of completely sequenced Thermotogae strains based on shared core genes was performed. It appeared that Petrotoga mobilis DSM 10674(T), originally isolated from a North Sea oil-production well, is the closest relative of D. tunisiensis L3. Comparative genome analyses of P. mobilis DSM 10674(T) and D. tunisiensis L3 showed …

Complete genome sequence of the hydrogenotrophic, methanogenic archaeon Methanoculleus bourgensis strain MS2(T), Isolated from a sewage sludge digester.

ABSTRACT Methanoculleus bourgensis , of the order Methanomicrobiales , is a dominant methanogenic archaeon in many biogas-producing reactor systems fed with renewable primary products. It is capable of synthesizing methane via the hydrogenotrophic pathway utilizing hydrogen and carbon dioxide or formate as the substrates. Here we report the complete and finished genome sequence of M. bourgensis strain MS2 T , isolated from a sewage sludge digester.

Differential enumeration and in situ localization of microorganisms in the hindgut of the lower termite Mastotermes darwiniensis by hybridization with rRNA-targeted probes

We examined the abundance and spatial distribution of major phylogenetic groups of the domain Bacteria in hindguts of the Australian lower termite Mastotermes darwiniensis by using in situ hybridization with group-specific, fluorescently labeled, rRNA-targeted oligonucleotide probes. Between 32.0 +/- 7.2% and 52.3 +/- 8.2% of the DAPI-stained cells in different hindgut fractions were detected with probe EUB338, specific for members of the domain Bacteria. About 85% of the prokaryotic cells were associated with the flagellates of the thin-walled anterior region (P3a) and the thick wall of the posterior region (P3b/P4) of the hindgut, as shown by DAPI staining. At most, half of the EUB338-det…

Bacillus species in the intestine of termites and other soil invertebrates

Soil invertebrates harbour a complex microbial community in their intestinal system. The total number of microbes in the hindgut of soil invertebrates can reach a titre of 10(11) ml(-1). The gut microbes play an indispensable role in the digestion of food and are of ecological importance in the global carbon cycle. The gut microbiota can include a variety of micro-organisms from the three domains Bacteria, Archaea and Eucarya. The bacterial groups from the intestinal systems are mainly affiliated to the proteobacteria, the gram-positive groups Firmicutes and Actinobacteria, the Bacteroides/Flavobacterium branch and the spirochetes. The Archaea are represented by methanogens. The eukaryotic …

Mikrobielle Synthese von Biopolymeren aus nachwachsenden Rohstoffen

Isolation of Desulfovibrio intestinalis sp. nov. from the hindgut' of the lower termite Mastotermes darwiniensis.

A Gram-negative, anaerobic sulfate-reducing bacterium was isolated from hindgut contents of the lower termite Mastotermes darwiniensis Froggatt (strain KMS2). Strain KMS2 is motile by a single polar flagellum. The isolate possesses desulfoviridin and catalase activity. The G+C content of its DNA is in the range of 54.5-55.5 mol% (strain KMS2). It respires hydrogen and different low molecular weight organic compounds in the presence of sulfate, thiosulfate, and sulfite, and also oxygen. The isolated strain ferments pyruvate. Fastest growth with a doubling time of 12.5 h was obtained at 37°C and not at 28°C, the temperature at which the termites were grown. The isolate showed a 16S rDNA seque…

Coriobacteriia class. nov.

Identification of the ectosymbiotic bacteria of Mixotricha paradoxa involved in movement symbiosis

Mixotricha paradoxa, a trichomonad from the hindgut of the Australian termite Mastotermes darwiniensis Froggatt, is a rare example of a movement symbiosis between eukaryotic and prokaryotic microorganisms. The surface of Mixotricha paradoxa is covered with spirochaetes and a rod-shaped bacterium. The four flagella at the anterior end seem only to alter the direction of movement, while the ectosymbiotic spirochaetes propel the flagellate cells. Based on a 16S rDNA sequence analysis after a semi-specific PCR, and subsequent fluorescence in situ hybridization applying helper oligonucleotides and a denaturing step of the 16S rRNA, three different spirochaete clones could be clearly identified o…

Intestinal Spirochetes of Termites

Spirochetes differ from all other bacteria by their unique morphology and ­mechanism of motility. The cells possess a helical shape, and the flagella (axial filaments) are located in the periplasmic space. The flagella are attached to the cell poles and wrapped around the protoplasmic cylinder. The flagella and the protoplasmic cylinder are surrounded by a multilayered outer sheath or outer cell envelope (Canale-Parola, 1984). The 16S rRNA sequences demonstrated that the spirochetes represent a monophyletic phylum within the bacteria (Paster and Dewhirst, 2001).

Species-specific identification of Dekkera/Brettanomyces yeasts by fluorescently labeled DNA probes targeting the 26S rRNA.

Sequencing of the complete 26S rRNA genes of all Dekkera/Brettanomyces species colonizing different beverages revealed the potential for a specific primer and probe design to support diagnostic PCR approaches and FISH. By analysis of the complete 26S rRNA genes of all five currently known Dekkera/Brettanomyces species (Dekkera bruxellensis, D. anomala, Brettanomyces custersianus, B. nanus and B. naardenensis), several regions with high nucleotide sequence variability yet distinct from the D1/D2 domains were identified. FISH species-specific probes targeting the 26S rRNA gene's most variable regions were designed. Accessibility of probe targets for hybridization was facilitated by the constr…

Aerobic and facultatively anaerobic cellulolytic bacteria from the gut of the termite Zootermopsis angusticollis.

Aims: To demonstrate the occurrence of cellulolytic bacteria in the termite Zootermopsis angusticollis. Methods and Results: Applying aerobic cultivation conditions we isolated 119 cellulolytic strains from the gut of Z. angusticollis, which were assigned to 23 groups of aerobic, facultatively anaerobic or microaerophilic cellulolytic bacteria. 16S rDNA restriction fragment pattern and partial 16S rDNA sequence analysis, as well as numerical taxonomy, were used for the assignment of the isolates. The Gram-positive bacteria of the actinomycetes branch could be assigned to the order Actinomycetales including the genera Cellulomonas/Oerskovia, Microbacterium and Kocuria. The Gram-positive bact…

Lactic Acid Bacteria

A typical lactic acid bacterium grown under standard conditions is aerotolerant, acid tolerant, organotrophic, and a strictly fermentative rod or coccus, producing lactic acid as a major end product. It lacks cytochromes and is unable to synthesize porphyrins. Its features can vary under certain conditions. Catalase and cytochromes may be formed in the presence of hemes and lactic acid can be further metabolized, resulting in lower lactic acid concentrations. Cell division occurs in one plane, except pediococci. The cells are usually nonmotile. They have a requirement for complex growth factors such as vitamins and amino acids. An unequivocal definition of LAB is not possible (Axelsson, Lac…

Effect of carboxymethyl cellulose on tartrate salt, protein and colour stability of red wine

Background and Aims Recent studies have confirmed a long-term effect of carboxymethyl cellulose (CMC) for tartrate salt stabilisation in white wine. It has been argued that CMC is not only less effective in red wine but also interacts with proteins and polyphenols generating turbidity and change in colour. In order to explain these effects, we studied in detail the impact of CMC on haze formation and colour stability of red wine. Methods and Results The influence of CMC concentration was tested with ten samples of red wine produced from several grape cultivars. The haze-forming material was analysed by sodium dodecyl-sulfate polyacrylamide electrophoresis and the protein composition by high…

Psychrotolerant Sulfate-reducing Bacteria from an Oxic Freshwater Sediment Description of Desulfovibrio cuneatus sp. nov. and Desulfovibrio litoralis sp. nov.

The most abundant culturable sulfate-reducing bacteria were isolated from the littoral sediment of the oligotrophic Lake Stechlin. The strains STL1 and STL4 were obtained from the oxic uppermost layer, while strain STL6 was isolated from the anoxic zone in 20 to 30 mm depth. The isolates showed a striking morphological feature in tapering off at one end of the cell. Physiological characteristics related them to the genus Desulfovibrio. They contained desulfoviridin. H2, formate, pyruvate, lactate, and fumarate were utilized with sulfate, sulfite, thiosulfate, or elemental sulfur as electron acceptors. All isolates were able to reduce oxygen and survived 120 h of aeration. However, aerobic g…

Microbial production of biopolymers from the renewable resource wheat straw.

Aims Production of poly-s-hydroxybutyrate (PHB) and the chemical basic compound lactate from the agricultural crop ‘wheat straw’ as a renewable carbon resource. Methods and Results A thermal pressure hydrolysis procedure for the breakdown of wheat straw was applied. By this means, the wheat straw was converted into a partially solubilized hemicellulosic fraction, consisting of sugar monomers, and an insoluble cellulosic fraction, containing cellulose, lignin and a small portion of hemicellulose. The insoluble cellulosic fraction was further hydrolysed by commercial enzymes in monomers. The production of PHB from the sugar monomers originating from hemicellulose or cellulose was achieved by …

Application of MALDI-TOF-MS and nested SAPD-PCR for discrimination ofOenococcus oeniisolates at the strain level

Oenococcus oeni is the most important lactic acid bacterium during vinification and is frequently used as a commercial starter culture for malolactic fermentation. Since different strains exhibit a high heterogeneity concerning wine-related physiological characteristics, reliable methods for their differentiation at the strain level are needed, especially for the development of starter cultures. Microbial identification by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has increased over the last few years. In the present study, we used MALDI-TOF-MS for the differentiation of 11 O. oeni isolates originating from 5 different wine-growing reg…

Polysaccharide‐containing Cell‐wall Polymers of Archaea

Introduction Historical Outline Chemical Structure Glutaminylglycan Heteropolysaccharide Methanochondroitin Pseudomurein S-Layer Lipoglycan Occurrence Functions Biochemistry Biosynthesis Biological Activity Molecular Genetics Biodegradation Methanochondroitin Pseudomurein Production (Producers, World market, Applications, Patents) Outlook and Perspectives Acknowledgements Keywords: glutaminylglycan; heteropolysaccharide; methanochondroitin; pseudomurein; S-layer (glyco-)protein; Archaea; cell envelope; cell-wall polysaccharides

Studies on Two Exoenzymes Which Lyse Wine-Spoiling Bacteria

Microorganisms play an important role in the conversion of grape juice into wine. Different species of yeast are mainly responsible for the production of ethanol. Lactic acid bacteria also occur regularly in must or wine. They are mostly undesirable due to their capacity to produce wine-spoiling compounds. Especially strains of Lactobacillus brevis are able to produce biogenic amines as well as precursors of ethyl carbamate and different off-flavours (N-heterocycles, volatile phenols). By excessive formation of acetic acid some lactobacilli may even induce slow/stuck grape juice fermentations. In conventional winemaking, additions of sulphite or lysozyme are used to inhibit the growth of sp…

Isolation of acetic, propionic and butyric acid-forming bacteria from biogas plants.

In this study, acetic, propionic and butyric acid-forming bacteria were isolated from thermophilic and mesophilic biogas plants (BGP) located in Germany. The fermenters were fed with maize silage and cattle or swine manure. Furthermore, pressurized laboratory fermenters digesting maize silage were sampled. Enrichment cultures for the isolation of acid-forming bacteria were grown in minimal medium supplemented with one of the following carbon sources: Na(+)-dl-lactate, succinate, ethanol, glycerol, glucose or a mixture of amino acids. These substrates could be converted by the isolates to acetic, propionic or butyric acid. In total, 49 isolates were obtained, which belonged to the phyla Firm…

Ultrastructure and Organization of the Cytoskeleton in Oxymonas, an Intestinal Flagellate of Termites

ABSTRACT. Oxymonas has the characteristic structures and organization of other oxymonads including two separated pairs of basal bodies/flagella, a preaxostylar lamina, a paracrystalline axostyle, and an absence of mitochondria and Golgi. Like other Oxymonadinae genera it possesses a long proboscis, the rostellum which is terminated by the holdfast. Like the genera Pyrsonympha and Streblomastix, Oxymonas possesses a holdfast which permits it to attach to the cuticle of the termite hind-gut. This holdfast is subdivided into rhizoids and is filled with microfilaments. The rostellum is variable in length and contains two distinct microtubular bundles. One bundle is composed of convoluted microt…

Fast protocols for the 5S rDNA and ITS-2 based identification ofOenococcus oeni

To identify specific marker sequences for the rapid identification of Oenococcus oeni, we sequenced the 23S-5S internal transcribed spacer (ITS-2) region and the 5S rDNA of five different O. oeni strains and three phylogenetically related lactic acid bacteria (LAB). Comparative analysis revealed 100% identity among the ITS-2 region of the O. oeni strains and remarkable differences in length and sequence compared to related LAB. These results enabled us to develop a primer set for a rapid PCR-identification of O. oeni within three hours. Moreover, the comparison of the 5S rDNA sequences and the highly conserved secondary structure provided the template for the design of three fluorescence-la…

Analysis of propionate‐degrading consortia from agricultural biogas plants

Abstract In order to investigate the propionate‐degrading community of agricultural biogas plants, four propionate‐degrading consortia (Ap1a, N12, G12, and Wp2a) were established from different biogas plants which were fed with renewable resources. The consortia were cultivated in a batch for a period of 2–4 years and then analyzed in an 8‐week batch experiment for microbial succession during propionate degradation. Community shifts showed considerable propagation of Syntrophobacter sulfatireducens, Cryptanaerobacter sp./Pelotomaculum sp., and “Candidatus Cloacamonas sp.” in the course of decreasing propionate concentration. Methanogenic species belonged mainly to the genera Methanosarcina,…

The yeastWickerhamomyces anomalusAS1 secretes a multifunctional exo-β-1,3-glucanase with implications for winemaking

A multifunctional exo-β-1,3-glucanase (WaExg2) was purified from the culture supernatant of the yeast Wickerhamomyces anomalus AS1. The enzyme was identified by mass spectroscopic analysis of tryptic peptide fragments and the encoding gene WaEXG2 was sequenced. The latter codes for a protein of 427 amino acids, beginning with a probable signal peptide (17 aa) for secretion. The mature protein has a molecular mass of 47 456 Da with a calculated pI of 4.84. The somewhat higher mass of the protein in SDS–PAGE might be due to bound carbohydrates. Presumptive disulphide bridges confer a high compactness to the molecule. This explains the apparent smaller molecular mass (35 kDa) of the native enz…

Cell Envelopes of Methanogens

Methanogens play an important role in the global carbon cycle, because they are involved in the last step of anaerobic degradation of organic material to methane. Although the first report on methane emanation from aquatic muds was given by Alessandro Volta in the year 1776, the first methanogen was not obtained in pure culture before 1947. Special culture techniques had to be developed for growing the strict anaerobic methanogenic isolates. The methanogens were the first species of the archaeal domain (Archaea) detected. Their unique biochemical and genetic properties have stimulated basic investigations of this microbial group in the last three decades. The methanogenic Archaea possess a …

Primary Structure of Selected Archaeal Mesophilic and Extremely Thermophilic Outer Surface Layer Proteins

The archaea are recognized as a separate third domain of life together with the bacteria and eucarya. The archaea include the methanogens, extreme halophiles, thermoplasmas, Sulfate reducers and Sulfur metabolizing thermophiles, which thrive in different habitats such as anaerobic niches, salt lakes, and marine hydrothermals systems and continental solfataras. Many of these habitats represent extreme environments in respect to temperature, osmotic pressure and pH-values and remind on the conditions of the early earth. The cell envelope structures were one of the first biochemical characteristics of archaea studied in detail. The most common archaeal cell envelope is composed of a single cry…

Manganese-dependent growth of Oenococci

Abstract Oenococci are usually grown in complex media supplemented with tomato juice. The manganese concentration of tomato juice is about 10 µM, which shows a growth-stimulating effect. Unexpectedly, we found that high concentrations of manganese (34 mM) could replace tomato juice. At this concentration, far exceeding the concentration in growth media, several Oenococcus oeni strains yielded the same cell density as in the presence of tomato juice. The observed significant differences in the manganese dependence should also have an impact on the growth of oenococci in must and wine, where they are used as starter cultures for the removal of malic acid.

Molecular organization of selected prokaryotic S-layer proteins.

Regular crystalline surface layers (S-layers) are widespread among prokaryotes and probably represent the earliest cell wall structures. S-layer genes have been found in approximately 400 different species of the prokaryotic domains bacteria and archaea. S-layers usually consist of a single (glyco-)protein species with molecular masses ranging from about 40 to 200 kDa that form lattices of oblique, tetragonal, or hexagonal architecture. The primary sequen ces of hyperthermophilic archaeal species exhibit some characteristic signatures. Further adaptations to their specific environments occur by various post-translational modifications, such as linkage of glycans, lipids, phosphate, and sulf…

Fast identification of wine related lactic acid bacteria by multiplex PCR

The microflora of must and wine consists of yeasts, acetic acid bacteria and lactic acid bacteria (LAB). The latter group plays an important role for wine quality. The malolactic fermentation carried out by LAB leads to deacidification and stabilisation of wines. Nevertheless, LAB are often associated with wine spoilage. They are mainly responsible for the formation of biogenic amines. Furthermore, some strains produce exopolysaccharide slimes, acetic acid, diacetyl and other off-flavours. In this context a better monitoring of the vinification process is crucial to improve wine quality. Moreover, a lot of biodiversity studies would also profit from a fast and reliable identification method…

Biotic methylation of mercury by intestinal and sulfate-reducing bacteria and their potential role in mercury accumulation in the tissue of the soil-living Eisenia foetida

Abstract Monomethylmercury as one of the most toxic mercury species influences the health and development of higher organisms and tends to accumulate in the tissue of animals and humans. The aim of this study was to explore the mercury methylating capability of (1) intestinal microbiota of the soil-living earthworm Eisenia foetida (E. foetida) and (2) intestinal sulfate reducing-bacteria in pure cultures. After exposing animals to inorganic mercury chloride (4 mg kg−1 Hg2+) in soil and sterile soil for ten days, the amount of methylmercury in tissue was measured. Despite sterilization of soil, the accumulation of the organic mercury species in tissue was 51 ng g−1. To elucidate the potentia…

III. Biochemistry of S-layers

During evolution prokaryotes have developed different envelope structures exterior to the cell wall proper. Among these surface components are regularly arranged S-layers and capsules. The structural characterization and the detailed chemical analysis of these surface molecules is a prerequisite to understand their biosynthesis and functional role(s) at the molecular level. Of particular interest are the glycosylated S-layer proteins which belong to the first prokaryotic glycoproteins ever described. Their characterization was performed on strains belonging to the thermophilic Bacillaceae and included structural studies and experiments to learn about the pathways for the glycan biosynthesis…

Complete genome sequence of the hydrogenotrophic Archaeon Methanobacterium sp Mb1 isolated from a production-scale biogas plant

Methanobacterium sp. Mb1, a hydrogenotrophic methanogenic Archaeon, was isolated from a rural biogas plant producing methane-rich biogas from maize silage and cattle manure in Germany. Here we report the complete genome sequence of the novel methanogenic isolate Methanobacterium sp. Mb1 harboring a 2,029,766 bp circular chromosome featuring a GC content of 39.74%. The genome encodes two rRNA operons, 41 tRNA genes and 2021 coding sequences and represents the smallest genome currently known within the genus Methanobacterium. (C) 2013 Elsevier B.V. All rights reserved.

Production of methyl mercury in the gut of the Australian termite Mastotermes darwiniensis

Animals are often exposed to or can ingest heavy metals along with their food. Therefore, we tested whether the hindgut microbiota of Mastotermes darwiniensis possesses the capability to form methyl mercury. The termite M. darwiniensis (Isoptera) was fed with saw dust containing different concentrations of inorganic mercury. Methyl mercury was determined by purge-and-trap capillary gas chromatography-atom fluorescence spectrometry (CGC-AFS) using ethyl mercury chloride as the internal standard. Total mercury concentrations were determined in the termite tissue by inductive coupled plasma-isotope dilution mass spectrometry (ICP-IDMS) after microwave-assisted digestion. The obtained results s…

Complete genome sequence of the methanogenic neotype strain Methanobacterium formicicum MF(T.).

The neotype strain Methanobacterium formicicum MFT (DSM1535), a hydrogenotrophic methanogenic Archaeon, was isolated from a domestic sewage sludge digestor in Urbana (IL, USA). Here, the complete genome sequence of the methanogen is reported. The genome is 2,478,074 bp in size, featuring a GC content of 41.23%. M. formicicum MFT encodes several genes predicted to be involved in adaptation to abiotic stress such as high osmolarity. The strain MFT is of biotechnological importance since M. formicicum strains are often found in production-scale biogas plants and it is suggested as a starter culture for the anaerobic biomethanation process. (C) 2014 Elsevier B.V. All rights reserved.

Influence of epigallocatechin gallate and phenolic compounds from green tea on the growth of Oenococcus oeni

Aims:  To investigate the effect of phenolic compounds on the growth of Oenococcus oeni. Methods and Results:  Oenococci are usually grown in media often supplemented with complex additives such as tomato juice. In order to improve our knowledge about the growth requirements of oenococci, we added several juices and leaf extracts such as green tea to the culture media and screened them for growth-stimulating substances to substitute complex supplements such as juices by more defined components. We found that also green tea could cause a growth stimulation of Oenococcus oeni strain B2. Conclusions:  Further experiments showed that the stimulating effect was as a result of the phenolic compou…

Micromanipulation Techniques for the Isolation of Single Microorganisms

A prerequisite for the biochemical and physiological investigation of microorganisms is the isolation and management of pure cultures. The only absolute criterion of purity for a bacterial culture is that it has been derived from the progeny of a single cell. Failure to apply this criterion may lead to much effort in proving the purity of a culture. All strains upon which research is to be based should therefore be rigorously purified before starting to investigate the properties of individual organisms (Johnstone 1969). Ecologically oriented microbiologists are faced especially with the problem of how to obtain a pure culture of certain microbial strains from their densely populated natura…

Cellulose Digestion in the Termite Gut